As a final commentary on native bromodomain interactions beyond acetylated
histones, acetylated transcription factor-bromodomain interactions are relevant
for controlling gene expression. Disruption of such interactions can also be targets
for drug discovery. Several examples in the literature have included the CBP
bromodomain with acetylated p53 [36] and CREB [34], the PCAF bromodomain
with acetylated TAT [37], and the BRD4 bromodomain interaction with acetylated
NF-κB [38], TWIST [39], and the androgen receptor [40]. In the case of the tandem
bromodomains of BRD4, it is thought that the predominant role of the first
N-terminal bromodomain is for histone recognition, while the second bromodomain
interacts with transcription factors [35]. However, this may not be the mechanism in
all cases.
3 Part II: Early Small-Molecule Bromodomain Inhibitor
Discovery
3.1 PCAF
The bromodomain of the p300/CBP-associated factor protein, PCAF, holds
historical precedent as the first bromodomain for which a small-molecule inhibitor
was developed. Until 2005, the functional consequences of inhibiting a human
bromodomain were untested due to a lack of small-molecule inhibitors. At this
time, Zhou and co-workers described the first reported bromodomain inhibitor,
NP1 (Fig. 1), targeting the PCAF bromodomain and demonstrated selectivity over
two other bromodomains from CBP and TIF1β [16]. The authors were motivated by
the fact that acetylation of the viral transcriptional activator, TAT, at K50 was
essential for transcription of essential genes for the HIV life cycle through interaction
with the PCAF bromodomain. Blocking such an interaction could then suppress
viral replication. Using a 2D
1 H15 N HSQC NMR screen of several thousand
compounds, the authors identified several molecules which bound outside the
histone binding site, targeting the BC and ZA loops. Structure activity studies in
an ELISA format using a biotinylated TAT peptide led to NP1 whose binding was
further confirmed via an NMR solution structure of the complex (PDBID: 1WUG).
Hu et al. would slightly improve on this affinity using a higher-throughput fluorescence polarization assay with a related analog leading to inhibition constants at
submicromolar levels (IC 50 ¼ 0.93 μM) versus an IC 50 of 1.37 μM for NP1 in the
same assay [41]. They also demonstrated preliminary anti-HIV cellular activity data.
Since the original work by Zhou and co-workers, several high-affinity inhibitors
of PCAF are available with high selectivity against human bromodomains. In
addition to HIV infection, aberrant PCAF function has been linked to cancer and
neuroinflammation. As such, these inhibitors should be of broad use to the community. One such inhibitor based on a triazolophtalazine scaffold, L-45 or L-Moses
(Fig. 4), was reported by Moustakin et al. in 2017 to be cellular active and highly
298
W. C. K. Pomerantz et al.
histones, acetylated transcription factor-bromodomain interactions are relevant
for controlling gene expression. Disruption of such interactions can also be targets
for drug discovery. Several examples in the literature have included the CBP
bromodomain with acetylated p53 [36] and CREB [34], the PCAF bromodomain
with acetylated TAT [37], and the BRD4 bromodomain interaction with acetylated
NF-κB [38], TWIST [39], and the androgen receptor [40]. In the case of the tandem
bromodomains of BRD4, it is thought that the predominant role of the first
N-terminal bromodomain is for histone recognition, while the second bromodomain
interacts with transcription factors [35]. However, this may not be the mechanism in
all cases.
3 Part II: Early Small-Molecule Bromodomain Inhibitor
Discovery
3.1 PCAF
The bromodomain of the p300/CBP-associated factor protein, PCAF, holds
historical precedent as the first bromodomain for which a small-molecule inhibitor
was developed. Until 2005, the functional consequences of inhibiting a human
bromodomain were untested due to a lack of small-molecule inhibitors. At this
time, Zhou and co-workers described the first reported bromodomain inhibitor,
NP1 (Fig. 1), targeting the PCAF bromodomain and demonstrated selectivity over
two other bromodomains from CBP and TIF1β [16]. The authors were motivated by
the fact that acetylation of the viral transcriptional activator, TAT, at K50 was
essential for transcription of essential genes for the HIV life cycle through interaction
with the PCAF bromodomain. Blocking such an interaction could then suppress
viral replication. Using a 2D
1 H15 N HSQC NMR screen of several thousand
compounds, the authors identified several molecules which bound outside the
histone binding site, targeting the BC and ZA loops. Structure activity studies in
an ELISA format using a biotinylated TAT peptide led to NP1 whose binding was
further confirmed via an NMR solution structure of the complex (PDBID: 1WUG).
Hu et al. would slightly improve on this affinity using a higher-throughput fluorescence polarization assay with a related analog leading to inhibition constants at
submicromolar levels (IC 50 ¼ 0.93 μM) versus an IC 50 of 1.37 μM for NP1 in the
same assay [41]. They also demonstrated preliminary anti-HIV cellular activity data.
Since the original work by Zhou and co-workers, several high-affinity inhibitors
of PCAF are available with high selectivity against human bromodomains. In
addition to HIV infection, aberrant PCAF function has been linked to cancer and
neuroinflammation. As such, these inhibitors should be of broad use to the community. One such inhibitor based on a triazolophtalazine scaffold, L-45 or L-Moses
(Fig. 4), was reported by Moustakin et al. in 2017 to be cellular active and highly
298
W. C. K. Pomerantz et al.